1. Caspase-dependent and -independent pathways in neuronal apoptosis after spinal cord injury Kay L.H. Wu, Chin Hsu and Julie Y.H. Chan Graduate Institute of Medicine, Kaohsiung Medical university, and Department o Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan Introduction 1.Spinal cord injury (SCI) results in irreversible neuronal damage leading to apoptotic cell death. 2. Accumulated evidence indicates that mitochondria initiate apoptosis via cellular events that include caspases-dependent and caspases-independent pathways. 3. We study the hypotheses that mitochondrial respiratory impairment promotes neuronal apoptosis and the impairment is induced via early stage of free radical production after SCI. Aims of Study 1. To identify the temporal profiles of apoptosis in the spinal cord after injury. 2. To delineate the relationship between mitochondrial respiratory dysfunction and neuronal apoptosis after spinal cord injury. 3. To investigate the involvement of free radical in mitochondrial dysfunction in the spinal cord after injury. Materials and Methods 1 52 34 7 10 Tissue fixation Electromicroscopy Genomic DNA Proteins Mitochondria DNA Laddering Western Blot T8T8 14 Summary and Conclusion Free radical Neuron Nucleus AIF DNA fragmentation Caspase-3 activation Apoptosis PARP activation I II III IV Caspase-9 activation cyt. c Figure 1. DNA fragmentation detected in the spinal cord after T8 transection (SCT) alone or with addition of coenzyme Q10 (CoQ 10) treatment. Values are mean  SEM, n = 9-12 animals per group. *P<0.05 vs. baseline control (C) in the Dunnett multiple-range test, or # P<0.05 vs. SCT groups in the Scheffé multiple-range test. M C 1 2 3 4 5 7 10 14 SCT only SCT with CoQ10 Figure 3. Time-course of changes in the mitochondrial respiratory complex I activity, ATP production in the spinal cord after SCT alone or with addition of CoQ10 treatment. Values are mean  SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or # P<0.05 vs. SCT groups in the Scheffé multiple-range test. Also shown is the representative electron microscopic photomicrographs illustrating swelling of the mitochondrial cristae after SCT. Normal control (50000 X) Post SCT 1 day (50000 X) Mitochondria complex I activity in the spinal cord after injury ATP concentration in the spinal cord after injury Figure 5. Time-course changes of iNOS expression or NOx levels in the spinal cord after SCI alone or with addition of CoQ10 treatment. Values are mean  SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or # P<0.05 vs. SCT groups in the Scheffé multiple-range test. Also shown is the representative photomicrographs illustrating the distribution of dihydroethidium or expression of NADPH oxidase subunits in the injured spinal cord. Cytosolic iNOS expression in the spinal cord after injury NOx concentration in the spinal cord after injury gp91 phox p47 phox p67 phox C 1 2 3 4 5 7 10 14 Dihydroethidium stain in 3rd day after SCI Nucleus AIF expression (NeuN, AIF and TUNEL triple-labeled cells, 1000 X) 3 rd day after SCI NeuNAIF TUNEL Merge Figure 4. Time-course of changes in the expression of apoptosis inducing factor (AIF) in the spinal cord after SCT alone or with addition of CoQ10 treatment. Values are mean  SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or # P<0.05 vs. SCT groups in the Scheffé multiple-range test. Also shown is the representative photomicrographs illustrating co-localization of AIF-immunoreactivity with NeuN-immunoreactivity in the TUNEL-positive cells. 0 1 2 3 4 5 7 10 14 Figure 2. Time-course of changes in the expression of caspase-dependent cytosolic proteins in the spinal cord after SCT alone or with addition of CoQ10 treatment. Values are mean  SEM, n = 9-12 animals per group. *P<0.05 vs. control (0) in the Dunnett multiple-range test, or # P<0.05 vs. SCT groups in the Scheffé multiple-range test. Cytosolic cleaved caspase-9 expression Cytosolic cleaved caspase-3 expression Cytosolic cytochrome c expression Cleaved PARP expression C 1 2 3 4 5 7 10 14